The ability to clone and express products of interest such as recombinant peptides and proteins in large amounts has become increasingly important. The ability to purify high levels of proteins is important in the human pharmaceutical and biotechnological field, for example for producing protein pharmaceuticals as well as in the basic research setting, for example for crystallizing proteins to allow the determination of their three dimensional structure. Proteins that are otherwise difficult to obtain in quantity can be over-expressed in a host cell and subsequently isolated and purified.
The choice of an expression system for the production of recombinant proteins depends on many factors, including cell growth characteristics, expression levels, intracellular and extracellular expression, post-translational modifications and biological activity of the protein of interest, as well as regulatory issues and economic considerations in the production of therapeutic proteins. Key advantages of mammalian cells over other expression systems such as bacteria or yeast are the ability to carry out proper protein folding, complex N-linked glycosylation and authentic O-linked glycosylation, as well as a broad spectrum of other post-translational modifications. Due to the described advantages, eukaryotic and in particular mammalian cells are currently the expression system of choice for producing complex therapeutic proteins such as monoclonal antibodies.
The most common approach to obtain high expressing host cells (also called high producers) generates an appropriate expression vector for expressing the product of interest as a first step. The expression vector drives the expression of the polynucleotide encoding the product of interest in the host cell and provides at least one selectable marker for generating the recombinant cell line. Key elements of mammalian expression vectors usually include a constitutive or inducible promoter capable of robust transcriptional activity; optimized mRNA processing and translational signals that usually include a Kozak sequence, a translation termination codon, mRNA cleavage and polyadenylation signals, a transcription terminator and selectable markers for the preparation of stable cell lines and for gene amplification; furthermore a prokaryotic origin of replication and selectable markers for vector propagation in bacteria can be provided by the expression vector.
In recent years the focus of development was concentrating on the design of improved vectors for gene expression in host cells. Despite of the plethora of available vectors, however, robust polypeptide/protein production with a high yield in mammalian cells is still challenging.
One established procedure for obtaining high producing cell lines expressing the product of interest with high yield is the stable transfection of the host cells. However, the stable integration into the genome is a rare event and only a small subset of stably transfected cells are high producers. Their selection is accordingly challenging.
Selectable markers and selection systems are widely used in genetic engineering, recombinant DNA technology and the production of recombinant products in order to obtain host cells expressing the product of interest with high yield. Respective systems are also useful to generate and identify stably transfected clones. The primary goal of using respective selectable markers and selection systems is to introduce a selectable gene which upon exposure to selective growth conditions allows the identification of cells capable of high-level production of the introduced selectable marker and accordingly, the recombinant product of interest. Increasing the yield of product expression can be e.g. achieved by gene amplification using cells lines e.g. deficient in an enzyme such as dihydrofolate reductase (DHFR) or glutamine synthetase (GS) in conjunction with expression vectors containing genes encoding these selectable marker enzymes and agents such as methotrexate (MTX), which inhibits DHFR, and methionine sulfoxamine (MSX) which inhibits GS.
One prominent selection system which is commonly used in the prior art is the dihydrofolate reductase/MTX selection system. Dihydrofolate reductase (DHFR) catalyzes the NADP-dependent reduction of dihydrofolic acid to tetrahydrofolic acid (THF). THF is then intraconverted to 10-formyl-DHF and 5,10-methylene-DHF which are used in the de novo biosynthesis of purines and thymidylate, respectively. DHF is the byproduct of the catalytic activity of thymidylate synthase (TS) which catalyzes the convertion of dUMP to dTMP in a 5,10-methylene-THF dependent reaction. Thus, DHFR is crucial for the recycling of THF cofactors that are essential for the biosynthesis of purine and pyrimidine nucleotides that are neccassary for the DNA replication. Hence, cells (for example CHO cells) that lack the DHFR gene (i.e. by targeted genomic deletion) can be used as recipients for the transfection of the DHFR gene in a medium that is free of nucleotides. After transfection, the cells can be subjected to gradual increase in the concentrations of the antifolate MTX, a most potent DHFR inhibitor (Kd=1 pM), thereby forcing the cells to produce increased levels of DHFR. After multiple rounds of selection, the selectable marker DHFR frequently undergoes significant amplification. Also more sensitive mutant forms of the respective selectable markers can be used in conjunction with wildtype host cells. Alternatively, a mutant mouse DHFR with a major resistance, i.e. less sensitivity, to MTX or other mutant forms of DHFR has also been extensively used as a dominant selectable marker that markley enhanced the acquisition of high level MTX-resistance in transfected cells. However, a major disadvantage of the DHFR/MTX selection system used in the prior art is that this technique utilizes a mutagenic cytotoxic agent, MTX, that can particularly in higher concentrations alter the genotype of the recipients cells. This frequently results in MIX-resistant cell populations in which no expression of the target gene of interest is present due to loss of function mutations for example in the reduced folate carrier (RFC)/or loss of RFC gene expression, both of which abolish MTX uptake. However, increasing/high concentrations of MTX are necessary, in order to achieve sufficiently stringent selection conditions in order to isolate host cells producing the product of interest with a sufficient yield.
As becomes apparent, a high stringency selection system is crucial to enrich high producing cells from a transfected population. The higher the stringency of the selection system the lower the number of low producers after the selection process and the higher the chance to find the very rare ultra high producing clones in a transfected cell population.
Therefore, it is the object of the present invention to provide a stringent selection system for selecting host cells producing a product of interest with high yield, as well as methods for producing a product of interest with sufficient yield. In particular, it is the object of the present invention to provide a stringent selection system which requires less amounts of toxic agents, in particular MTX. Furthermore, it is the object of the present invention to provide a method for producing a product of interest with a high yield.